PROJECT 4: PROJECT SUMMARY Prostate cancer (PC) is the most commonly diagnosed non-skin cancer in American men. Androgen deprivation therapies (ADT) that target the androgen receptor (AR) are the mainstay treatment for metastatic PC. New- generation anti-androgen enzalutamide (Enz) has been effective in prolonging patient life for almost a full year and yet resistance develops rapidly. Importantly, castration-resistant PC (CRPC) is driven primarily by aberrant activation of the AR in the milieu of low androgen. The AR exerts its tumorigenic roles mainly through genomic regulation of target gene expression. This genomic action is tightly regulated by a number of cofactors, one of which is FOXA1. FOXA1 is traditionally known as an AR co-activator in androgen-stimulated AR signaling. However, biomarker studies have reported controversial results regarding FOXA1 as a positive or negative outcome predictor, suggesting context- or treatment-dependent roles. In addition, recurrent, loss-of-function mutations of FOXA1 have been found in PC indicating tumor suppressive roles. We recently found that although FOXA1 is slightly up-regulated in localized PC relative to benign tissues, it is dramatically down-regulated in CRPC. Mechanistic studies revealed that FOXA1 has dual roles in its regulation of AR signaling. In the presence of androgen, FOXA1 enhances AR signaling and induces cell growth. However, in the absence of androgen, FOXA1 suppresses residual AR activity and as such FOXA1 loss leads to androgen-independent AR activation and castration-resistant PC cell growth. Moreover, we found that FOXA1 loss also turns on TGF /slug pathway contributing to cell migration and invasion. Further, TGF /slug pathway is also activated in Enz-resistant CRPC cells. We thus hypothesize that clinically-available ALK5 inhibitors that target TGF /slug signaling may suppress CRPC progression and overcome Enz-resistance in combinatorial therapies. To test this hypothesis, three specific aims are proposed. In Aim 1 we will elucidate the roles of FOXA1 in suppressing CRPC progression in vitro and most importantly in xenograft tumors in vivo. Aim 2 will characterize the downstream pathways of FOXA1 in CRPC cells and PC specimens. In Aim 3 we will investigate the efficacy of Enz in combination with ALK5 inhibitor in CRPC cell lines and xenografts, whereas Aim 4 will develop a phase I clinical trial of combinatorial Enz and ALK5 inhibitor treatment of metastatic CRPC. For Aims 1 and 2, we present preliminary data, but in LNCaP cells that are not castrate resistant; we have yet to test our hypothesis in CRPC cell lines, which are more relevant to this proposal. In addition, the proposed animal experiments described in Aim 1 and IHC staining in Aim 2 involve a substantial amount work and have not yet been performed. Furthermore, virtually all of the proposed work in Aims 3 and 4 is yet to be done. SPORE funding is critical for us to carry out this project.